Method and device for diagnosis using an oscillating airflow

ABSTRACT

It has surprisingly been found that nasal NO release is greatly increased by the presence of a an oscillating airflow in healthy subjects. Accordingly, a device comprising means for generating and/or maintaining an oscillating airflow is described and suggested for use in the diagnosis of a condition of the upper airways of a human. The invention also encompasses method wherein the concentration and/or flow of at least one gaseous component in nasally exhaled air is determined and recorded in the presence and absence of an oscillating airflow acting on the upper airways or parts thereof.

This application is a U.S. national phase application of InternationalPCT/SE03/00175, filed Jan. 31, 2003, which claims priority to SwedishPatent Application No. 0200300-2 filed Feb. 1, 2002, all of which arehereby incorporated by reference in their entirety.

The present invention relates to the investigation of the upper airwaysof a human, and in particular to a device and a method for use in thedetermination of the function and status of the sinuses, as well as foruse as an aid in the diagnosis of various diseases of the human upperairways.

BACKGROUND OF THE INVENTION

The paranasal sinuses are cavities inside the facial bones surroundingthe nose, which communicate with the nasal cavity through narrow ostia.The mucous membrane of the sinus is continuous with that of the nasalcavity. The maxillary sinuses constitute the largest of these cavities.An opening in the medial wall of the maxillary sinus communicates withthe middle meatus of the nose. Obstruction of the sinus ostium is acentral event in the pathogenesis of sinusitis.

The gas nitric oxide (NO) is released in the human respiratory tract.The major part of the NO found in exhaled air originates in the nasalairways and this NO can be measured non-invasively with differentsampling techniques. It is known that a large production of NO takesplace in the parnasal sinuses where an inducible NO synthase isconstantly expressed in the epithelial cells. NO is released also fromother sources in the nose e.g. the nasal mucosa. However, the relativecontribution from the different NO sources in the upper airways to theNO found in nasal air is difficult to estimate. The sinuses communicatewith the nasal cavity through ostia and the rate of gas exchange betweenthese cavities is dependent e.g. on the size of the ostia A patentostium is essential for maintenance of sinus integrity. Blockage of theostium, e.g. caused by virus-induced mucosal swelling, results indecreased oxygen tension, mucosal edema, reduced mucociliary transportand eventually bacterial colonisation. Earlier studies have shown thatnasal NO levels are markedly decreased in airway disorders affecting thesinuses, e.g. primary ciliary dyskinesia (PCD) and cystic fibrosis (CF).The concentrations of NO in the healthy sinuses are very high, sometimesmore than 20 ppm.

Sinusitis is a very common disease causing much human suffering andenormous costs for the society. The self-reported prevalence of chronicsinusitis in the USA is about 12% of the population. There are severalproblems involved in the diagnosis and treatment of this disorder. Forexample, headache, rhinorrhea and nasal congestion are extremely common,but these symptoms do not necessarily imply sinusitis. Therefore thetrue incidence of sinusitis is lower.

Proper ventilation of the sinuses is essential for sinus integrity. Infact, occlusion of the ostia is considered the key factor in thepathogenesis of sinusitis. Such occlusion may be of mechanical ormucosal origin i.e. septum deviation, nasal polyposis, allergic rhinitisor most commonly an acute viral infection. The basic principles oftreatment are to cure any infection present and to promote sinusdrainage both during and after treatment to prevent recurrence.Antibiotics remain the cornerstone treatment in medical handling ofacute infectious sinusitis. In addition, medical intervention with nasaldecongestants as well as surgical treatment is frequently used inprevention and treatment of chronic sinusitis with the purpose ofimproving sinus drainage.

PRIOR ART

U.S. Pat. No. 6,142,952 describes a method and apparatus for detectionand diagnosis of airway obstruction. The invention of the '952 patent isdirected towards pressure and flow data measurements of pressurizedbreathable gas supplied to the patient's airways with an oscillationcomponent or forced probing signal. The gas is supplied with aninterface including a mask and a flexible hose and the oscillatingcomponent or forced probing signal with a loudspeaker coupled to afrequency generator. Pressure and flow of the breathable gas in theinterface are measured or sampled to obtain characteristics of thepatient's airway.

An experimental model for the study of gas exchange through the ostiumof the maxillary sinus has been developed (Aust, R., and Drettner, B.,Uppsala J Med Sci, 79; 177-186, 1974). In their article, Aust andDrettner first refer to previously known methods involving theintroduction of a small electrode for pO₂-measurements into the maxlarysinus and a continuous recording of the oxygen content in the sinus.Aust and Drettner instead developed an experimental model using a rubbernose, moulded from a cadaver, and a nitrogen filled syringe representingthe maxillary sinus. An air stream through the nasal model was generatedby a respirator, and the pressure changes in the syringe were recorded.As a measure of the gas exchange taking place, the oxygen content in thesyringe was measured. The respiratory frequency was constant in allmodel experiments, whereas the volume of the syringe and the diameter atthe connection between the nasal model and the syringe could be varied.The volume of the syringe represented the volume of the maxillary sinus,and the diameter at the connection the diameter of the ostia The resultsindicated that the gas exchange is dependent of the diameter of theostia.

In another article (Aust, R. and Drettner, B., Acta Otolaryng78:432-435, 1974) a method for measuring the functional size of themaxillary ostium in living patients is described. The method is based onthe measurement of the pressure rise in a maxillary ostium with patentostium caused by an air stream led into the sinus through a cannulaentering the antrum through the lower nasal meatus.

Still earlier methods for studying the patency of the maxillary ostiumrelied on the recording of the pressure in both the nasal canal and themaxillary sinus during breathing, blowing and sniffing.

A noninvasive test is the 133-xenon washout technique in which a mixtureof air and 133-xenon is insufflated into the nasal cavities (Paulsson etal., Ann. Otol. Rhinol. Laryngol., 2001; 110:667-74). The passage intothe sinuses is facilitated by increasing pressure obtained by thesubjects inflating a balloon. The washout of 133-xenon is monitored by ascintillation camera allowing single photon emission computed tomography(SPECT). The washout halftimes are used as a measure of the ventilationof the sinuses.

The invasive tests are potentially painful for the patients andcumbersome to perform. They are therefore not suitable for use in dailyclinical practice. A easy non-invasive test that could be used tomeasure sinus ostial patency would be most useful. Such test could helpto identify subjects at risk of developing sinusitis. Also, it could beused to monitor effects of surgical or medical interventions aimed forprevention of sinusitis.

SUMMARY OF THE INVENTION

The above problems are solved by a device according to the presentinvention, for use in the analysis of the condition of the upper airwaysof a human, in particular the condition of the sinus or sinuses of ahuman, wherein said device comprises means for generating and/ormaintaining an oscillating airflow acting on the upper airways or partsthereof and is suitable for connection to means for determining andrecording the concentration and/or flow of a gas present in the exhaledair of the human. Another aspect of the invention is a method for use asan aid in the investigation of the upper airways of a human, inparticular for the analysis of the condition of the sinus or sinuses ofa human, wherein the concentration and/or flow of at least one gaseouscomponent in nasally exhaled air is determined and recorded in thepresence and absence of an oscillating airflow. Further features andassociated advantages of the present invention will be evident from thedescription, examples and claims, incorporated herein by reference.

SHORT DESCRIPTION OF THE DRAWINGS

The invention will be disclosed in closer detail in the followingdescription, and examples, with reference to the attached drawings inwhich

FIG. 1 shows an original tracing of NO during a single breath nasalexhalation with humming (a) or silently (b);

FIG. 2 shows the effect of repeated humming maneuvers on nasal NOoutput. Five consecutive exhalations with humming were performed at 5seconds intervals. A progressive reduction in NO levels was observedafter each maneuver until a plateau was reached;

FIG. 3 shows the nasal NO output measured during a silent exhalation atbaseline, immediately after repeated silent nasal exhalations andimmediately after repeated humming maneuvers (five consecutive 10 snasal exhalations with humming). *=p=0.002 compared to baseline, n=6;

FIG. 4 shows the change in nasal NO output (%) after topical nasalapplication of an NO synthase inhibitor (L-NAME). The subjects exhaledeither silently or with humming. (*p=0.002, n=6);

FIG. 5 shows the effect of silent nasal exhalation or humming on nasalNO output in controls and patients with nasal polyposis.

FIG. 6 is a schematic presentation of an experimental model of the sinus(G), the ostium (syringe tip) and the nasal cavity (C). A denotes theflow/pressure meter, B denotes the flow resistor, D denotes the soundgenerator (rubber duck call), E denotes the display, and F denotes theNO analyzer.

FIG. 7 shows the influence of ostium size on sinus gas exchange in theexperimental model. A subject performed a single breath exhalation at afixed flow rate (0.2 L/sec) either silently (unfilled) or with phonation(filled). Sinus gas exchange was calculated by measuring NO in thesyringe before and immediately after each exhalation;

FIG. 8 shows the effect of three different humming frequencies on NOlevels in a sinus/nasal model. A subject exhaled orally in the model ata fixed flow rate of 0.2 L/s, a NO concentration of 8 ppm, a resistanceof 1 cmH₂O and an ostium size of 1.9 mm.

DESCRIPTION

The present inventors have surprisingly found that nasal NO release isgreatly increased by the presence of an oscillating airflow in healthysubjects. This increase likely reflects an increased contribution of NOfrom the paranasal sinuses. Measurement of nasal NO in the presence ofan oscillating airflow is an easy non-invasive test that can givevaluable information about the condition of the upper airways, e.g. theNO production in the sinuses as well as sinus ostial function.

An external source of an oscillating airflow or a humming soundgenerated by the human under study causes the air to oscillate which inturn seems to increase the exchange of air between the sinuses and thenasal cavity. This was also confirmed in the two-compartment modelsystem where an oscillating airflow simulating that achieved by hummingresulted in a great increase in NO levels. The volume in the syringe,the NO concentration as well as the diameter of the syringe tip(representing the sinus ostium) were chosen to resemble physiologicalvalues for these parameters. The normal size of an ostium isapproximately 2.4 mm. Interestingly, NO levels were found to be stronglydependent on the diameter of the syringe tip. This shows that theincrease in nasal NO during in vivo humming or in the presence of anoscillating airflow is dependent on ostial size. In addition, sinus NOconcentrations and humming frequency were shown to affect sinusventilation. It is possible that several other factors will influencethe rate of exchange between the two cavities.

Surprisingly, topical administration of a NOS inhibitor in the nosereduced nasal NO output by more than 50% during quiet breathing but hadno effect on the increase in nasal NO during humming. Again, thissupports the notion that this increase is due to enhanced ventilation ofthe sinuses, which are not reached by topical nasal sprays. The presentinventors cannot exclude that humming increases NO release also fromother sources within the nose. For example the oscillating airflows maygenerally increase the release of NO solved in the epithelial cells andfluid linings. However, as shown in the experiments below, a suggestedmethod could be to start with repeated humming maneuvers to empty thesinuses, immediately followed by a silent nasal exhalation along with NOmeasurements. In this way sinus contribution to nasally exhaled NO wouldbe minimized which could help to unmask changes in nasal mucosal NOoutput.

Based on the above findings, confirmed in both in vivo studies, and inan experimental set-up, the inventors make available a device for use inthe analysis of the upper airways, and in particular for determining thecondition of the sinus or sinuses of a human, wherein said devicecomprises means for generating and/or maintaining an oscillating airflowhaving a frequency, duration and flow sufficient to enhance theventilation of the sinuses, said device being suitable for connection tomeans for determining and recording the concentration and/or flow of agas present in the exhaled air of said human being. In one embodiment ofthe invention the gas is endogenous NO.

In this context it is understood that the term “upper airways” refers tothe airways located above the vocal cords, including the paranasalsinuses, nasal cavity, nasopharynx, epipharynx, oropharynx, oral cavityand the hypopharynx.

In this context it is understood that the term “nasal airways” refers tothe airways extending from the nostrils to the nasopharynx.

In this context it is understood that the term “endogenous gas” refersto gas produced within the body of a human.

In this context it is understood that the condition of the upper airwaysencompasses the absence or presence of one of the following: aninflammatory condition, an airway infection, common-cold, tumors, drugrelated effects, anatomical abnormalities, the patency of the sinusostia, the size of the sinuses, the biochemical status of the sinuses,sinusitis affecting one or more sinuses, the location said sinusitis,the risk for developing sinusitis, the bacteriological status of thesinuses, or a combination thereof in particular, the device is useful inthe diagnosis of the conditions of the sinuses, e.g. the presence orabsence of a pathological condition affecting the sinuses, and itslocation, such as primary cilliary dyskinesia (PCD) and cystic fibrosis(CF), nasal polyposis, allergic rhinitis, an inflammatory condition ofthe upper airways, a common cold, or a combination thereof.

According to one embodiment of the invention, the means for generatingand/or maintaining an oscillating airflow sufficient to enhance theventilation of the sinuses is a means for electronically or mechanicallygenerating said airflow.

Preferably, said device is adapted to unilateral measurements, that isthe measurement of the effect of an oscillating airflow on nasallyexhaled gases, in one nostril at the time. This can be achieved eitherby using nasal olives, occluding one nostril while the measurement isperformed in the other, or when using a mask, by occlusion of onenostril at the time.

According to another embodiment, the means for generating and/ormaintaining an oscillating airflow is a means for registering theduration, frequency and/or volume of an oscillating airflow produced bythe patient, e.g. by humming, comprising a feed-back mechanism, such asmeans for indicating to said patient that a pre-set necessary duration,frequency, flow and/or volume has been reached.

According to an embodiment of the present invention the oscillatingairflow oscillates at a frequency between 1 to 1000 Hz, preferablybetween 10 to 1000 Hz, more preferably between 100 to 1000 Hz, and mostpreferably between 100 to 500 Hz.

According to another embodiment of the present invention the oscillatingairflow oscillates at a frequency close to the resonance frequency ofthe paranasal sinuses.

According to another embodiment of the present invention the airflow isaspirated from the upper airways and the aspirator coupled to a soundgenerator that causes the air to oscillate.

According to a preferred embodiment of the present invention, saiddevice is suitable for connection to a device for determining andrecording the concentration and/or flow of a first gas present in theexhaled air of a human being, and a device for supplying a secondbreathing gas devoid of or containing minute amounts and/or a knownamount of said first gas to said human being. Most preferably said firstgas is nitric oxide and said second gas is a nitric oxide-free gassuitable for inhalation. One example of such a device is the NIOX®NO-analyser (Aerocrine AB, Solna, Sweden).

It is also possible to determine the concentration and/or flow of othergases, endogenous or exogenous. Examples of such gases include nitricoxide (NO), nitrogen, oxygen, carbon dioxide, carbon monoxide, andsuitable inert gases, e.g. argon or xenon.

A further embodiment involves means for analysis of the timedistribution of the NO, i.e. the shape of the curve when theconcentration is plotted in relation to time and/or flow. This analysiswill give information on the dynamic behavior of the sinuses and it iscontemplated that this will make it possible to distinguish betweendegree and type of congestion, sinus volume, diameter of ostia, and toelucidate the possible pathology underlying the sinus problems underinvestigation in each particular patient The absolute amount of exhaledgas, e.g. NO, and its change over time, such as the increase, decrease,associated peak, slope and plateau values will reveal anatomical andphysical differences. Using patient data collected from a number ofpatients, or data collected from measurements performed on the samepatient at different occasions or using different frequency, flow andduration of the oscillating airflow, additional or more preciseinformation can be gained. The following anatomical and physicaldifferences are likely to be revealed using this approach:

-   -   ostium size/patency    -   sinus volume    -   sinus NO-production    -   NO-absorption

The present invention also makes available a method for the analysis ofthe condition of the upper airways of a human, in particular the sinusor sinuses of a human, wherein the concentration and/or flow of at leastone gaseous component in nasally exhaled air is determined and recordedin the presence and absence of an oscillating airflow.

According to an embodiment of the present invention the oscillatingairflow in the method of analysis oscillates at a frequency between 1 to1000 Hz, preferably between 10 to 1000 Hz, more preferably between 100to 1000 Hz, and most preferably between 100 to 500 Hz.

According to another embodiment of the present invention the oscillatingairflow in the method of analysis oscillates at a frequency close to theresonance frequency of the paranasal sinuses.

According to another embodiment of the present invention the airflow inthe method of analysis is aspirated from the upper airways and theaspirator coupled to a sound generator that causes the air to oscillate.

According to an embodiment of this method, the condition of the upperairways encompasses the absence or presence of one of the following: aninflammatory condition, an airway infection, common cold, tumors, drugrelated effects, anatomical abnormalities, the patency of the sinusostia, the size of the sinuses, the biochemical status of the sinuses,sinusitis affecting one or more sinuses, the location said sinusitis,the risk for developing sinusitis, the bacteriological status of thesinuses, or a combination thereof. The present method is particularlysuitable for determining the condition of the sinuses, such as thepresence of absence of a pathological condition affecting the sinuses,such as primary ciliary dyskinesia (PCD) and cystic fibrosis (CF), nasalpolyposis, allergic rhinitis, an inflammatory condition of the upperairways, or a combination thereof. The present invention thus makesavailable methods for use in the diagnosis of any one of these orrelated conditions/diseases.

According to another embodiment of this method, the condition of theupper airways is determined at at least two occasions, before and afterthe administration of a drug or the performance of a therapeuticintervention and the result is used to evaluate the effect of said drugor intervention.

The oscillating airflow necessary for the measurement can be achieved byurging the person undergoing the test or investigation, to produce anoscillating airflow simulating that achieved by humming. In that case,it may be necessary to include a feed-back function, i.e. to registerthe duration, frequency, flow and/or volume of this oscillating airflowproduced by the patient, and indicating to said patient when a pre-setnecessary duration, frequency, flow and/or volume has been reached.

The oscillating airflow can also be artificially produced and directedto the upper airways or a part thereof, e.g. to the sinus or sinuses.

According to one embodiment of the inventive method, the concentrationand/or flow of a first gas present in the exhaled air of a human beingis determined, while a second breathing gas devoid of or containing aminute and/or known amount of said first gas is, supplied to said humanbeing. Preferably said first gas is nitric oxide and said second gas isa nitric oxide-free gas suitable for inhalation. One example of a devicesuitable for performing these functions is the NIOX® NO-analyser(Aerocrine AB, Solna, Sweden).

The method may also involve the determination of the concentrationand/or flow of other gases, endogenous or exogenous. Examples of suchgases include nitric oxide (NO), nitrogen, oxygen, carbon dioxide,carbon monoxide, and suitable inert gases, e.g. argon.

A further embodiment involves a step of analyzing the time distributionof the NO, i.e. the shape of the curve when the concentration is plottedin relation to time and/or flow. This analysis will give information onthe dynamic behavior of the sinuses and it is contemplated that thiswill make it possible to distinguish between degree and type ofcongestion, sinus volume, diameter of ostia, and to elucidate thepossible pathology underlying the sinus problems under investigation ineach particular patient. The absolute amount of exhaled gas, e.g. NO,and its change over time, such as the increase, decrease, associatedpeak, slope and plateau values will reveal anatomical and physicaldifferences. Using patient data collected from a number of patients, ordata collected from measurements performed on the same patient atdifferent occasions or using different frequency, flow and duration ofthe oscillating airflow, additional or more precise information can begained. The following anatomical and physical differences are likely tobe revealed using this approach:

-   -   ostium size (patency)    -   sinus volume    -   sinus NO-production    -   NO-absorption

A comparison of curves obtained when using the two-compartment model, invivo measurements in the presence of an oscillating airflow, andmeasurements during silent nasal exhalation has been made. It is seenthat, in the two-compartment model, the concentration of NO decreases asno new NO is produced in the syringe simulating the sinus (results notshown). In the in vivo tests, the curve exhibits a similar increase andpeak (FIG. 1), although these values vary between the subjects tested.The decrease is however less, due to the replenishment of NO in thesinuses, and both the slope of the curve as well as the plateau levelreached varies between the subjects tested.

This test has the obvious advantage of being non-invasive, rapid andobjective. It will also be of interest to study if measurements duringhumming can be used to gain more information on conditions affecting theupper airways, and e.g. to better separate patients with nasal disordersand altered nasal NO release from healthy controls. Such disordersinclude e.g. CF, PCD, nasal polyposis and allergic rhinitis.

EXAMPLES 1. In Vivo Measurements 1.1 Healthy Controls

Characterization of Nasal NO During Humming

Ten healthy non-smoking volunteers (age 25-47 years, 6 males,) withoutany history of allergy, nasal disease, asthma or any other chronic lungconditions were recruited. Airway NO output was measured with achemiluminescence system (NIOX®, Aerocrine AB, Stockholm, Sweden)designed to meet the ATS guidelines for exhaled NO (American ThoraicSociety. Am J Respir Crit Care Med 1999; 160:2104-17). The analyzer wascalibrated with standard gas mixtures of NO (987 parts per billion, AGAAB, Sweden). NO levels were measured during oral and nasal single breathexhalations. A tight fitting mask covering the nose was used for nasalmeasurements, and a mouthpiece was used for oral exhalations. Thesubjects started each maneuver by inhaling NO-free air through the noseand then exhaled at a fixed flow rate (0.20 L/s) for ten seconds eitherquietly or with nasal humming or oral phonation. The fixed flow rate wasachieved by a dynamic flow restrictor in the analyzing system combinedwith a computerized visual feed back display of flow. The dynamic flowrestrictor uses an elastic membrane valve to mechanically adjust flowrate and keep exhalation at 0.20 L/s within a wide range of exhalationpressures with minimal variation.

Nasal NO output during humming was calculated by subtracting the valuesobtained during silent nasal exhalations as described earlier (Lundberget al., Thorax 1999; 54:947-952; Palm et al., Eur Resp J 2000;16:236-41). NO release was calculated as the mean output (nl/min) duringthe last 80% of the exhalation.

To investigate if humming could exhaust the source of NO the subjectsperformed five consecutive humming maneuvers with different timeintervals (5 seconds, 1 minute and 3 minutes) between each humming.Also, repeated silent nasal exhalations were performed at 5 secintervals. Based on the results obtained from consecutive hummingmaneuvers (see below), all other humming in this study were preceded bya 3 minute period of silence.

Effects of NO Synthase Inhibition

In six of the subjects baseline nasal and oral NO measurements were madeboth during humming and silent exhalations. Then either a solution ofNG-L-arginine methyl ester (L-NAME) (Sigma, Poole, UK.) 15 mg (2.2 mM)in 2.5 ml of saline or saline alone was delivered in random orderthrough both nostrils by a jet nebulizer (Devilbiss, Somerset, Pa., USA)and the NO measurements were repeated 20 min. after application of thesolutions.

Effects of Flow, Pressure and Frequency During Humming

To compare the results from the model described above to the in vivosituation we performed additional experiments in five of the subjects.They were asked to exhale in turn at two fixed flow rates (0.20 and 0.25L/s) against no resistance or at a resistance of 50 cm H₂O L⁻¹ s⁻¹ for aperiod of ten seconds either silently or with nasal humming or oralphonation. This was followed by nasal humming maneuvers at threedifferent sound frequencies. Frequency was registered with themicrophone taped on the neck of the subject. NO output was calculatedfrom the mean concentration during the entire exhalation.

Results

In all humming experiments an initial NO peak was observed followed by aprogressive decline (FIG. 1). Total nasal NO output increased duringhumming as compared to silent exhalation (from 471±73 nL/min duringsilent exhalation to 2233±467 nL/min during humming; p<0.001) (FIG. 1).Orally exhaled NO was 144±20 nL/min with silent exhalation and 152±20nL/min with phonation (p=0.22).

NO output measured during five single-breath humming maneuvers with 3minutes intervals between each humming was similar showing anintra-individual variability of less than 15%. With one minute intervalsthe intra individual variability was nearly 70%. With 5 secondsintervals NO decreased progressively after each maneuver until a stableplateau was reached at a level of 571±88 nL/min compared to levelsduring the first humming of 2233±467 nL/min, p=0.002 (FIG. 2). In allsubjects the low plateau was reached within four nasal hummingmaneuvers. In contrast, five consecutive silent nasal exhalations with 5sec intervals did not affect NO output (FIG. 3). However, silent nasalNO output measured immediately after repeated humming maneuvers waslower than basal silent NO in all subjects (261±35 nL/min vs 384±39nL/min; p=0.021). There was a substantial variability in the reductionof silent nasal NO after consecutive humming ranging between 5-50%.Topical application of L-NAME reduced silently exhaled nasal NO levelsby more than 50% from 392±33 nL/min to 194±24 nL/min; p=0.002 (FIG. 4).In contrast, the humming-induced increase in NO output was not affected(2417±894 nL/min before L-NAME vs 2368±811 nL/min after L-NAME, p=0.77).

Increasing the exhalation flow rate during humming from 0.20 to 0.25 L/sresulted in higher nasal NO output (from 807±172 to 1074±197 ni/min,p<0.05).

Change of humming frequency also affected nasal NO output. NO levelswere 940±77 nL/min at 130 Hz, 807±77 nL/min at 150 Hz and 719±58 nL/minat 450 Hz (p<0.05). NO output increased with higher nasal pressureduring humming (from 807±77 nL/min at 1 cm H2O to 932±26 nL/min at 10 cmH2O, p>0.05).

1.2 Sinus Volume

A preliminary study involving two healthy subjects, for whom the sinusvolumes were previously recorded, was performed. The level of nasallyexhaled NO was determined during silent breathing and during humming,using a standardised chemiluninescence system (NIOX®, Aerocrine AB,Solna, Stockholm) as above. The NO-values recorded during silentbreathing were approximately the same. The peak NO-value for the subjecthaving a larger sinus volume was however considerably higher (about 1500ppb) than for the subject having a smaller sinus volume (300 ppb). Inthe subject having larger sinus volume, the NO-value decreased moreslowly. The results indicate that an analysis of the peak, slope andplateau values can give information on anatomical and physiologicalfeatures of the upper airways, and in particular the sinuses. (Resultsnot shown.)

1.3 Correlation to Sinus Problems

Ten healthy non-smoking subjects (age 25-52 years, 5 males) without anyhistory of allergy or chronic airway disorder and 10 patients withchronic sinusitis and nasal polyposis (age 30-56 years, 5 males) tookpart in the study. None of the controls had any ongoing respiratorytract infection at the time of the study. The patients were on a waitinglist for sinus surgery. All had bilateral polyps and completely opaquesinuses according to a previous CT scan. All were on treatment withtopical nasal corticosteroids, three had concomitant asthma and four hadaspirin intolerance. NO was measured in nasal single-breath exhalationsusing a chemiluminescence system developed to meet the criteria of theATS guidelines for exhaled NO measurements (Aerocrine AB, Stockholm,Sweden). A tight fitting mask covering the nose was used and thesubjects exhaled nasally with closed mouth at a fixed flow rate (0.10l/s) for 10 s either silently or with humming. NO levels were calculatedas the mean output (nl/min) during the last 80% of the exhalation.Exhalation flow rate was monitored and variation was minimal (<0.02l/s).

Results

During quiet exhalation nasal NO was similar in controls and patients(189±27 nl/min vs 162±22 nl/min). Nasal NO increased 7-fold duringhumming (to 1285±189 nl/min) in controls but remained completelyunchanged in the patients (169±21 nl/min, FIG. 5).

It was shown that the increase in nasal NO during humming is completelyabsent in patients with nasal polyposis. The most likely explanation isa lack of air-passage between the sinuses and the nasal cavity.Interestingly, one of the patients had surgery during the course of thisstudy and in this patient nasal NO increased during humming to almostnormal levels two weeks after the operation (data not shown).

2. Two-compartment Model Study—the Sinus/Nasal Model

Description of the Model

NO output was measured in a two-compartment model resembling the nasalcavity and one sinus (FIG. 6). A syringe (representing the sinus) wasfilled with various NO gas concentrations ranging between 2 and 10 ppm(AGA AB, Sweden), and connected horizontally to a plastic cylinder(representing the nasal cavity) via a Luer fitting. The diameter of thesyringe tip (representing the ostium) was varied between 0.8 to 4.0 mm.The volume of the syringe was varied between 5 and 20 ml. The distal endof the cylinder (nasal cavity) was left open or connected to a HansRudolph resistor of 50 cm H₂O L⁻¹ s⁻¹ thereby generating cylinderpressures of either 1 or 10 cmH₂O. Flow and pressure were measured by alinear pneumotachymeter (Hans Rudolph Inc). Resulting NO levels weremeasured at the distal end of the cylinder by a rapid-responsechemiluminescence system (Aerocrine AB, Stockholm, Sweden). The signaloutput from these devices were connected to a computer-based system(Aerocrine NO system, Aerocrine AB, Stockholm, Sweden) and yielded aninstant on-screen display of flow, pressure, NO concentration and NOoutput.

Artificial Generation of Humming in the Model

Pressurized NO-free air was set to generate three different flow rates(0.20, 0.25 and 0.30 L/s). The air was led through the plastic cylinder(nasal cavity) either via a rubber duck call (Hudson & Co, UK), whichyielded a pulsating airflow or via a rubber duck call without the soundgenerating membrane (quiet control). Three duck calls with differentfundamental frequencies (120, 200 and 450 Hz) were used. NO was measuredduring a ten sec period and all experiments were repeated five times. Inan additional experiment, a turbulent flow was generated by leadingpressurized NO-free air through a plastic mesh connected to the cylinderand NO was measured as described above. This experiment was done withouta sound generating device.

In a separate experiment we studied the effect of 3 different hummingfrequencies (120, 200 and 450 Hz) on NO output from sinuses withdifferent resonance frequencies (120 or 200 Hz).

Human Humming in the Model

In the same model the pulsating airflow was also generated by a subjectperforming oral exhalation through the cylinder with or withoutphonation at two fixed flow rates (0.20 or 0.25 L/s) and three differentfrequencies (130, 150 or 450 Hz). NO output was calculated from theentire exhalation (10 sec) with subtraction of oral NO output. Allexperiments were repeated five times. To estimate the rate of airexchange between the two cavities, we also measured the remaining NOconcentration in the syringe at the end of each experiment.

Measurement of Artificial and Human Humming Sound Frequency.

The audio signal of humming was picked up by a TCM 110 Tiepin electretcondenser microphone placed on the plastic cylinder in the model (FIG.6) and recorded directly onto a PC by the Soundswell Signal Workstation.The fundamental frequency was extracted by its Corr module that computesthe autocorrelation of the audio signal in two adjacent time windows.The mean fundamental frequency and standard deviation were thendetermined by means of its histogram module.

The resonance frequency of the model system was calculated according toDurrant and Lovrinic (Bases of Hearing Science, 3^(rd) Ed., Williams andWilkins, Baltimore, 1995: 60).

Results

In the standard setting of the model we used a fixed flow rate of 0.2L/s, an NO concentration of 8 ppm, a pressure of 1 cm H₂O, a syringevolume of 15 ml, an ostium size of 1.9 mm and a humming frequency of 200Hz. The resonance frequency of this system was calculated to be 200 Hz.When changing one parameter in the experiments all other values werekept constant.

In all experiments using the model, artificial and human humming causedan increase in NO output compared to silent exhalation. When usingartificial humming in the model NO output increased >10-fold from23.7±0.1 nL/min during silent airflow to 295±4.5 nL/min during humming(p<0.05). When a subject was humming in the model NO output increasedfrom 27.7±0.1 nL/min during silent exhalation to 175±8 nL/min (p<0.05).No difference in NO output was seen in the model when using a turbulentflow compared to a non turbulent flow (25.2±0.2 nL/min and 23.7±0.1nL/min, respectively).

Effect of Ostium Size

Ostial diameters of 0.8, 1.29, 1.9, 2.1 and 4.0 mm were used. NO outputduring humming increased with larger ostium size (FIG. 7). With a ratiofor the ostium size of 1:1.6:2.4:2.6:5 the ratios for NO output in thehuman and artificial models were 1:4.5:6:14:30 and 1:8:13:15:39respectively. As an estimation of the rate of air exchange in the sinus,the remaining NO concentration in the syringe was measured immediatelyafter the exhalations (FIG. 7). We found no significant changes insyringe NO concentrations after silent exhalations regardless of ostiumsize. In contrast, during humming the air exchange was stronglydependent on ostium size and reached almost 100% with the largest ostium(FIG. 7).

Effect of Humming Frequency

We found significant changes in NO output by modifying the frequency ofhumming in all experiments. When using artificial humming in the modelNO output was 230±5.7 nL/min at a frequency of 120 Hz, 295±3.4 nL/min at200 Hz, and 143±2.0 nL/min at 450 Hz (p<0.05, FIG. 8).

In the human humming model NO levels were 204±11 nL/min at 130 Hz, 175±8nL/min at 150 Hz and 143±2 nL/min at 450 Hz (p<0.05, n=5). When studyingthe effect of different humming frequencies on NO output from syringeswith different resonance frequencies we found that the NO output wasgreatest when the humming frequency was close to the resonance frequencyof the particular sinus (Table I).

TABLE I Effect of humming frequency on NO output (nL/min) using sinuseswith different resonance frequency in the model. Sinus ResonanceFrequency Humming Frequency 120 Hz 200 Hz 120 Hz 1043 ± 10   527 ± 5.8200 Hz 561 ± 8.3 611 ± 7.7 400 Hz 286 ± 6.3 418 ± 8.1Effect of Syringe Volume

Table II and III show the result after humming when the ostial size, NOconcentration, flow and resistance were kept constant according to thestandard setting. Syringe volumes of 5, 10, 15 and 20 ml were used. Witha ratio for the sinus volumes of 1:2:3:4 the ratios for NO levels were1:2.5:5:7 in the artificial humming model and 1:2:4:5.5 in the humanhumming model.

Effect of Syringe NO Concentration

Table II and III show the effect of syringe NO concentration duringhumming. The NO concentrations of 2, 4, 8 and 10 ppm were used. With aratio for the syringe NO concentration of 1:2:4:5 the ratios for NOlevels in the artificial and human humming models were respectively1:2.1:4:5.5 and 1:2:3:7.

Effect of Airflow Rate

Results concerning NO output at different nasal flow rates duringhumming are shown in table II and III. With a ratio for the flow rate of1:1.25:1.5 the ratios for NO levels in the artificial and human hummingmodels were 1:1.25:1.4 and 1:1.5:2 respectively.

TABLE II The influence of sinus volume, sinus NO concentration and flowrate on resulting NO levels induced by an artificial pulsating airflowin a model of the nose and sinus (for details see methods). Artificialhumming NO output (nL/min) Sinus Volume 5 ml  79 ± 1.0 10 ml 159 ± 4.5*15 ml 295 ± 3.4* 20 ml 427 ± 3.7* NO concentration 2 ppm  76 ± 1.5 4 ppm162 ± 2.8* 8 ppm 295 ± 3.4* 10 ppm 434 ± ^(6.1)* Flow 0.20 L/sec 295 ±3.4 0.25 L/sec 369 ± 5.8* 0.30 L/sec 411 ± 7.6* *= p < 0.05

TABLE III The influence of sinus volume, sinus NO concentration and flowrate on resulting NO levels induced by human pulsating airflow in amodel of the nose and sinus (for details see methods). Human humming NOoutput (nL/min) Sinus Volume 5 ml  79 ± 1.0 10 ml  87 ± 3.6* 15 ml 175 ±8.0* 20 ml 242 ± 14.7* NO concentration 2 ppm  57 ± 8.1 4 ppm 118 ±14.6* 8 ppm 175 ± 8.0* 10 ppm 416 ± 32* Flow 0.20 L/sec 175 ± 8.0 0.25L/sec 268 ± 4.8* 0.30 L/sec 356 ± 10* *= p < 0.05Effect of Pressure

In the artificial humming model we found an increase in NO output withhigher pressure during humming (from 175±8 nL/min to 377±22 nL/min). Inthe human humming model we found a reduction as we increased thepressure (from 250±3.4 nL/min to 140±1.9 nL/min).

3. Statistics

The NO output was calculated for all sampling modalities as flow x NOconcentration. Non-parametric statistics with two-way p values wereused. For analysis of paired data Friedman's test and Wilcoxon's testwere used. A p value less than 0.05 was considered significant. Resultsare given as mean±SEM.

4. Discussion

The large and reproducible increase in nasal NO caused by humming inhealthy volunteers has been characterized, as well as in a model of thenose and sinus. The humming method gives relevant information about therelative contribution of NO from the nose and sinus as well as ostiumpatency. Several factors strongly indicate that the NO increase seenduring humming is due to a rapid washout of NO accumulated in theparanasal sinuses. The profiles of the nasal exhalation curves (peak andprogressive decline) in the model and in the human studies were verysimilar and the factors influencing NO levels were identical. Both thepeak and the total nasal NO output were markedly decreased followingrepeated consecutive humming manouvers and a complete recovery wasobserved after a 3 minute period of silence. Again, this pattern fitswell with the notion that humming empties the sinuses and that a periodof silence will allow for NO to accumulate again. A NOS inhibitor(L-NAME) applied locally in the nose reduced silent nasal NO levels by50% but had no effect on the increase during humming. Assuming that thisroute of administration mostly affects the nasal mucosa with a lesspenetration into sinuses, this also supports a sinus origin of nasal NOduring humming.

Ostium size seemed to be the most important factor affecting theincrease in nasal NO during humming. Sinus NO concentrations and hummingfrequency also affected sinus ventilation. Interestingly, the hummingfrequency affected sinus output both in the model and in the healthyvolunteers. These preliminary experiments have shown that theventilation of the sinus in the model is greatest when the hummingfrequency is close to the resonance frequency of the sinus model.

From the experiments looking at remaining NO in the syringe aftersingle-breath exhalations it is obvious that humming is an enormouslyeffective means of increasing sinus ventilation. This is also supportedby the in vivo experiments where the rapid decline in NO during hummingindicated sinus emptying. The results show that almost the entire sinusvolume is exchanged in one single exhalation if the subject is humming.Even when using a small ostial diameter humming was very effective inventilating the sinus in the model used. This suggests that hummingcould help to increase sinus ventilation in patients with sinusitis andpartly obstructed ostia.

In the present study silent nasal NO levels were between 5 to 50% lowerimmediately after repeated humming. If we assume that the sinuses areeffectively emptied by this maneuver, the decrease should fairy wellreflect the normal contribution from the sinuses to NO found in nasallyexhaled air. It is however important to note that this assumption may betrue only under the exact conditions of this study. Nevertheless usingthe methods described here, it may be possible to better separate sinusNO from nasal mucosal NO release.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

1. A device for use in diagnosis of a condition of the upper airways ina human, wherein said device comprises means for generating and/ormaintaining an oscillating airflow in said upper airways or partsthereof; said device being suitable for connection to means fordetermining and recording the concentration and/or flow of nitric oxide(NO) present in exhaled air of said human; and wherein the condition ofthe upper airways is an absence or presence of a condition selected fromthe group consisting of an inflammatory condition, an airway infection,a common cold, a tumor, a drug related effect, an anatomicalabnormality, a patency of the sinus ostia, a size of the sinuses, abiochemical status of the sinuses, a sinusitis affecting one or moresinuses, a location of said sinusitis, a risk for developing sinusitis,a bacteriological status of the sinuses, and a combination thereof.
 2. Adevice according to claim 1, wherein the means for generating and/ormaintaining an oscillating airflow comprises means for electronically ormechanically generating said airflow.
 3. A device according to claim 1,wherein the means for generating and/or maintaining an oscillatingairflow comprises means for registering a duration, frequency and/orvolume of an oscillating airflow produced by a patient coupled to ameans for indicating to said patient that a pre-set necessary duration,frequency and/or volume has been reached.
 4. A device according to claim1, wherein the oscillating airflow oscillates at a frequency between 1to 1000 Hz.
 5. A device according to claim 1, wherein the oscillatingairflow oscillates at a frequency between 10 to 1000 Hz.
 6. A deviceaccording to claim 1, wherein the oscillating airflow oscillates at afrequency between 100 to 1000 Hz.
 7. A device according to claim 1,wherein the oscillating airflow oscillates at a frequency between 100 to500 Hz.
 8. A device according to claim 1, wherein the oscillatingairflow oscillates at a frequency close to a resonance frequency of theparanasal sinuses.
 9. A device according to claim 1, wherein said devicecomprises means for unilaterally diagnosing an abnormal state affectingonly the right or left hand side of the nasal airways includingparanasal sinuses.
 10. A device for use in diagnosis of a condition ofthe upper airways in a human, wherein said device comprises means forgenerating and/or maintaining an oscillating airflow in said upperairways or parts thereof and means for determining and recording aconcentration and/or flow of nitric oxide (NO) present in exhaled air ofsaid human, and means for supplying a breathing gas devoid of orcontaining only minute and/or known amounts of NO to said human.
 11. Adevice according to claim 10, wherein said breathing gas is a nitricoxide-free gas suitable for inhalation.
 12. A device according to claim10, wherein said device is suitable for connection to means fordetermining and recording a concentration and/or flow of a tracer gas.13. A device according to claim 10, wherein said device is suitable forconnection to means for determining and recording the concentrationand/or flow of a tracer gas, said tracer gas being an endogenous gas.14. A device according to claim 10, wherein said device is suitable forconnection to means for determining and recording a concentration and/orflow of a tracer gas, said tracer gas being an exogenous gas.
 15. Amethod for diagnosing a condition of the upper airways in humancomprising: determining a concentration and/or a flow rate of at leastone gaseous component in a sample of nasally exhaled air in the presenceand absence of an oscillating airflow acting on the upper airways orparts thereof, wherein the condition of the upper airways is the absenceor presence of a condition selected from the group consisting of aninflammatory condition, an airway infection, a common cold, a tumor, adrug related effect, an anatomical abnormality, a patency of the sinusostia, a size of die sinuses, a biochemical status of the sinuses,sinusitis affecting one or more sinuses, a location of said sinusitis, arisk for developing sinusitis, a bacteriological status of the sinuses,or a combination thereof; and calculating the mean output of the gaseouscomponent.
 16. A method according to claim 15, wherein the oscillatingairflow oscillates at a frequency between 1 to 1000 Hz.
 17. A methodaccording to claim 15, wherein the oscillating airflow oscillates at afrequency between 10 to 1000 Hz.
 18. A method according to claim 15,wherein the oscillating airflow oscillates at a frequency between 100 to1000 Hz.
 19. A method according to claim 15, wherein the oscillatingairflow oscillates at a frequency between 100 to 500 Hz.
 20. A methodaccording to claim 15, wherein the oscillating airflow oscillates at afrequency close to a resonance frequency of sinus cavities.
 21. A methodaccording to claim 15, wherein said human is urged to produce theoscillating airflow simulating an oscillation achieved during humming.22. A method according to claim 15, wherein the oscillating airflow isartificially produced and directed to the upper airways of partsthereof.
 23. A method for diagnosing a condition of the upper airways ina human comprising: determining a concentration and/or a flow rate ofnitric oxide (NO) present in a sample of exhaled air by a human in thepresence and absence of an oscillating airflow acting on the upperairways or parts thereof, while a breathing gas devoid of or containingminute and/or known amounts of said NO has been supplied to said human;and calculating the mean output of NO.
 24. A method according to claim23, wherein said breathing gas is a nitric oxide-free gas suitable forinhalation.
 25. A method according to claim 23, wherein theconcentration and/or flow of an endogenous gas in nasally exhaled air isdetermined in the presence and in the absence of said oscillatingairflow acting on the upper airways.
 26. A method according to claim 23,wherein an exogenous gas is administered to the human and theconcentration and/or flow of said gas in nasally exhaled air isdetermined in the presence and in the absence of said oscillatingairflow acting on the upper airways.
 27. A method according to claim 23,wherein the condition of the upper airways is the absence or presence ofa condition selected from the group consisting of an inflammatorycondition, an airway infection, a common cold, a tumor, a drug relatedeffect, an anatomical abnormality, a patency of the sinus ostia, a sizeof the sinuses, a biochemical status of the sinuses, sinusitis affectingone or more sinuses, a location of said sinusitis, a risk for developingsinusitis, a bacteriological status of the sinuses, or a combinationthereof, and wherein the upper airways, nasal airways and paranasalsinuses are subjected to unilateral study by analyzing nasally exhaledair first from one nostril and then from the other nostril.
 28. A methodaccording to claim 15, wherein the condition of the upper airways isdetermined on at least two occasions, including before and afteradministration of a drug or performance of a therapeutic intervention inorder to evaluate the effect of said drug or intervention.
 29. A methodaccording to claim 24, wherein the condition of the upper airways isdetermined on at least two occasions, including before and afteradministration of a drug or performance of a therapeutic intervention inorder to evaluate the effect of said drug or intervention.